Glucose isomerases catalyze the reversible isomerization of glucose to fructose. Fructose is nowadays commonly applied as sugar substitute due to its higher sweetness compared to e.g. sucrose and glucose.
Many microorganisms are known to produce glucose isomerase, see for example the review articles by Wen-Pin Chen in Process Biochemistry, 15 June/July (1980) 30-41 and August/September (1980) 36-41, in which a large number of microorganisms, capable of producing glucose isomerase, are listed.
Several microorganisms can be used for the industrial production of glucose isomerases, among these Streptomyces Ampullariella and Actinoplanes are well known. The Wen-Pin Chen reference describes culture conditions for the microorganisms and recovery and purification methods for the produced glucose isomerases.
Generally the naturally occurring glucose isomerases also show a high affinity for sugars other than glucose. In this respect D-xylose, D-ribose, L-arabinose, D-allose and 6-deoxyglucose were found to be substrates of this enzyme. The K.sub.m values of D-glucose, D-xylose and D-ribose, were shown to vary from microorganism to microorganism and were reported to be in the range, of 0.086-0.920, 0.005-0.093 and 0.35-0.65M, respectively.
The K.sub.m values for xylose are significantly lower than for glucose, which implies that the correct name for the enzyme is in fact xylose isomerase. Furthermore, the V.sub.max of the commonly used glucose isomerases is higher on xylose than on glucose, which also suggest that xylose isomerase is a better name.
Since glucose isomerase is active on different substrates it may be advantageous to alter the substrate specificity depending on the desired reaction product, the specific process in which it is used or the wish to avoid unwanted side-products.
For the application of glucose isomerase in HFCS production a higher V.sub.max and a lower K.sub.m on glucose would be useful properties, since the reaction time and the enzyme costs would be reduced.
Another application of glucose isomerase is in the conversion of xylose to ethanol (Jeffries, T. W., Trends Biotechnol. 3 (1985) 208). A higher activity (V.sub.max) on xylose and/or a better affinity for xylose would be useful properties for this application.
The digestibility and the taste of feed for monogastric animals can be improved if glucose is converted enzymatically into fructose. In practice the application of glucose isomerase is hampered by the xylose isomerisation activity, which causes an unwanted formation of xylulose in feed. A glucose isomerase with no or reduced specificity (V.sub.max or K.sub.m) for xylose would be preferred for application in feed pretreatment.
Clearly, there is a need for altering the substrate specificity of glucose isomerases which would at the same time widen the field of the application of this enzyme.
Recently redesigning of the specific activity of enzymes with the aid of protein engineering techniques has been described. Wells et al. (Proc. Natl. Acad. Sci. U.S.A. 84 (1987) 5167) show an example for subtilisin. Bacillus licheniformis and B. amyloliquefaciens serine proteases differ by 31% (86 amino acid residues) in protein sequence and by a factor of 60 in catalytic efficiency on certain substrates. By substituting 3 of the 86 different amino acids from the B. amyloliquefaciens sequence by the corresponding B. licheniformis residues the catalytic activity of the mutant enzyme was improved nearly 60 fold.
In another paper it is described how a lactate dehydrogenase was changed into a malate dehydrogenase by mutating glutamine 102 into arginine 102 (Wilks et al., Science 242 (1988) 1541).
In both cases referred to above, serine protease and lactate dehydrogenase, the modification proposal was based on the comparison of the molecule to be modified and naturally occurring enzymes, which already showed the desired substrate specificity. In the same way the specificity of cytochrome p450.sub.15.alpha. was changed into the specificity of cytochrome p450.sub.coh by replacing Leu209 with Phe209 (Lindberg and Negishi, Nature 339 (1989) 632).
For glucose isomerase no naturally occurring enzyme is known, which shows a better specificity for glucose than for xylose. The above-mentioned method, based on the comparison of active sites of homologous enzymes having a different substrate specificity, can therefore not be applied to glucose isomerase.
WO 89/01520 (Cetus) lists a number of muteins of the xylose isomerase which may be obtained from Streptomyces rubiginosus and that may have an increased stability. The selection of possible sites that may be mutated is based on criteria differing from the ones used in the present invention. More than 300 mutants are listed but no data are presented concerning the characteristics and the alterations therein of the mutant enzyme molecules.